methods of behavior analysis in neuroscience - jerry j. buccafusco

Methods of
BEHAVIOR ANALYSIS
in NEUROSCIENCE

© 2001 by CRC Press LLC
METHODS & NEW FRONTIERS IN NEUROSCIENCE SERIES
Series Editors
Sidney A. Simon, Ph.D.
Miguel A.L. Nicolelis, M.D., Ph.D.
Published Titles
Apoptosis in Neurobiology
Yusef A. Hannun, Ph.D. and Rose-Mary Boustany, Ph.D.
Methods for Neural Ensemble Recordings
Miguel A.L. Nicolelis, M.D., Ph.D.

© 2001 by CRC Press LLC
Boca Raton London New York Washington, D.C.
CRC Press
Edited by
Jerry J. Buccafusco
Methods of
BEHAVIOR ANALYSIS
in NEUROSCIENCE

© 2001 by CRC Press LLC

Publisher/Acquiring Editor: Barbara Norwitz
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Library of Congress Cataloging-in-Publication Data

Methods of behavior analysis in neuroscience / edited by Jerry J. Buccafusco.
p. cm. — (Methods and new frontiers of neuroscience)
Includes bibliographical references and index.

ISBN 0-8493-0704-X (alk. paper)
1. Neurosciences—Handbooks, manuals, etc. 2. Animal behavior—Handbooks,
manuals, etc. 3. Laboratory animals—Psychology—Handbooks, manuals, etc. 4.
Behavioral assessment—Handbooks, manuals, etc. I. Buccafusco, Jerry J. II. Series.
[DNLM: 1. Behavior, Animal. 2. Neurosciences—methods. 3. Animals,
Laboratory—psychology. WL 100 M5925 2000]
RC343.M45 2000
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′

027—dc21 00-039762


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Series Preface

Our goal in creating the Methods & New Frontiers in Neuroscience Series is to
present the insights of experts on emerging experimental techniques and theoretical
concepts that are, or will be at the vanguard of Neuroscience. Books in the series
will cover topics ranging from methods to investigate apoptosis, to modern tech-
niques for neural ensemble recordings in behaving animals. The series will also
cover new and exciting multidisciplinary areas of brain research, such as computa-
tional neuroscience and neuroengineering, and will describe breakthroughs in clas-
sical fields like behavioral neuroscience. We want these to be the books every
neuroscientist will use in order to get acquainted with new methodologies in brain
research. These books can be given to graduate students and postdoctoral fellows

when they are looking for guidance to start a new line of research.
The series will consist of case-bound books of approximately 250 pages. Each
book will be edited by an expert and will consist of chapters written by the leaders
in a particular field. Books will be richly illustrated and contain comprehensive
bibliographies. Each chapter will provide substantial background material relevant
to the particular subject. Hence, these are not going to be only “ methods books.”
They will contain detailed “tricks of the trade” and information as to where these
methods can be safely applied. In addition, they will include information about
where to buy equipment, web sites that will be helpful in solving both practical and
theoretical problems, and special boxes in each chapter that will highlight topics
that need to be emphasized along with relevant references.
We are working with these goals in mind and hope that as the volumes become
available the effort put in by us, the publisher, the book editors, and individual
authors will contribute to the further development of brain research. The extent that
we achieve this goal will be determined by the utility of these books.

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Preface

Behavioral techniques used in animals to model human diseases and to predict the
clinical actions of novel drugs are as varied as the numbers of scientists who use
them. For behaviors as simple as locomotor performance in an open field, there are
dozens of experimental approaches and various components of movement that con-

tribute to overall motor activity. As behavioral models become more complex, there
is often a bewildering array of perturbations for a given task extant in the literature.
Yet behavioral analysis, as a tool for the basic neuroscientist, is becoming indis-
pensable as information gained at the molecular and cellular level is put into practice
in fully behaving animal subjects.
Since the neuroscientist trained in methodologies directed toward the molecular
and cellular level does not often have experience in the intricacies of animal behav-
ioral analyses, there is often much time devoted to assessing a complex literature,
or to developing an approach

de novo

. Specialists who are recognized experts in
several fields of cognitive and behavioral neuroscience have provided chapters that
focus on a particular behavioral model. Each author has analyzed the literature to
describe the most frequently used and accepted version of the model. Each chapter
includes: (1) a well-referenced introduction that covers the theory behind, and the
utility of the model; (2) a detailed and step-wise methodology; and (3) approach to
data interpretation. Many chapters also provide examples of actual experiments that
use the method.
The primary objective of the book is to provide a reference manual for use by
practicing scientists having various levels of experience who wish to use the most
well-studied behavioral approaches in animal subjects to better understand the effects
of disease, and to predict the effects of new therapeutic treatments on the human
cognition. In view of the large numbers of transgenic animals produced on an almost
daily basis, special attention is given to procedures designed for testing mice. While
there has been no attempt to cover all areas of animal behavior and sensory pro-
cessing, this text will help take the guesswork out of designing the methodology for
many of the most widely used animal behavioral approaches developed for the study
of brain disorders, drug abuse, toxicology, and cognitive drug development.

One cautionary note to the newcomer to the behavioral field is to not be deceived
by the manner in which this book presents its topics. As a matter of convenience,

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the topics have been arranged in chapter form, and there may be the false sense of
security that each method described is the last word on the subject. However, it is
often not sufficient to employ one of these methods to assess the cognitive status of
an animal. For example, when studying memory or recall, it is prudent to use a test
battery that can better provide a comfortable level of interpretation of the effect of
the perturbation applied to the subject. Spatial and non-spatial tasks should be
considered. If a negative reinforcer is involved, such as electrical shock, the animal
should be tested for his response to pain. Drugs or other manipulations that might
alter pain sensitivity could give false impressions in a shock-motivated memory task.
Drugs that affect motor activity may alter maze activity or swimming behavior, and
drugs that alter taste, appetite, or that induce GI disturbances could affect food-
motivated behaviors.
Whenever possible, the animal should be observed (at least initially) while
performing the task. It is often surprising to some investigators (this one included)
to find that the animal is using a technique to solve the problem posed to it that was
not considered in designing the task. A good example is the mediating or non-
mnemonic strategies that rats use to solve matching problems in various operant
paradigms. Most animals would rather use such strategies (such as orientating to a
proffered lever) to obtain food rewards than to use memory. Whenever possible, our
authors have provided some of these pitfalls in their chapters, although every possible
contingency could not be anticipated. Thus, it is in the best interest of the investigator
to use this book to help develop several strategies to understand the complex behav-
iors of animals as they respond to drugs, new diets, surgical interventions, or to

additional or fewer genes. While danger in anthropomorphizing the behavior of
animals always exists, the investigator should feel some level of confidence that
much of the behavioral literature is replete with instances of high predictive value
for similar perturbations in humans. Of course, species and strain differences can
limit such interpretations. Mice are clearly not little rats, and rats are not non-human
primates. Each species has a specific level of predictive value that should be assessed.
A final cautionary note is that investigators make every attempt to be as repro-
ducible as possible when studying animal behavior. Handlers, experimenters, food,
water, bedding, noise, surrounding visual cues, are just a few of the factors that
should be held constant when performing behavioral studies. Inconsistency contrib-
utes mightily to response variability in a population, and may even lead to a com-
pletely opposite behavior to the one expected.
At this point I would like to express my sincere thanks to the many authors
who contributed these chapters. Their difficult task in preparing this information
will make easier the tasks of our readers in their own efforts to assess animal
behavior. I would also like to acknowledge the support (moral and technical) of the
CRC staff, Publisher Barbara Norwitz, and Editorial Assistant Amy Ward, and the
Methods in Neuroscience Series Editors, Sidney Simon and Miguel Nicolelis.
Finally, I would like to thank my office administrator Vanessa Cherry for her many
contributions in getting this book together for publication, and to my wife Regina
Buccafusco, who is an education design specialist, for helping in the proofreading
of the chapters.

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The Editor

Jerry J. Buccafusco, Ph.D.

, is director of the Alzheimer’s Research Center, in the
Department of Pharmacology and Toxicology of the Medical College of Georgia.
He holds a joint appointment as Research Pharmacologist at the Department of
Veterans Affairs Medical Center. He holds the rank of Professor of Pharmacology
and Toxicology and Professor of Psychiatry and Health Behavior. Dr. Buccafusco
was trained classically as a chemist, receiving an MS degree in inorganic chemistry
from Canisius College in 1973. His pharmacological training was initiated at the
University of Medicine and Dentistry of New Jersey where he received a Ph.D.
degree in 1978. His doctoral thesis concerned the role of central cholinergic neurons
in mediating a hypertensive state in rats. Part of this work included the measurement
of several behavioral components of hypothalamically mediated escape behavior in
this model. His postdoctoral experience included two years at the Roche Institute
of Molecular Biology under the direction of Dr. Sydney Spector. In 1979 he joined
the Department of Pharmacology and Toxicology of the Medical College of Georgia.
In 1989 Dr. Buccafusco helped found and became the director of the Medical College
of Georgia, Alzheimer’s Research Center. The Center hosts several core facilities,
including the Animal Behavior Center, which houses over 30 young and aged rhesus
monkeys who participate in cognitive research studies.
Awards and honors resulting from Dr. Buccafusco’s research include the New
Investigator Award, National Institute on Drug Abuse, 1980; Sandoz Distinguished
Lecturer, 1983; Distinguished Faculty Award for the Basic Sciences, School of
Medicine, Medical College of Georgia, 1988; Callaway Foundation of Georgia,
Center Grant recipient, 1989; and the Distinguished Alumnus Award, University of
Medicine and Dentistry of New Jersey, 1998. Dr. Buccafusco also served as a
member of the Pharmacology II Study Section of the National Institute on Drug
abuse from 1989–1991, and he is a member the Scientific Advisory Board of the
Institute for the Study of Aging in New York.
Dr. Buccafusco holds memberships in several scientific societies. In his profes-
sional society, the American Society for Pharmacology and Experimental Therapeu-

tics, he serves as Chairman of the Graduate Student Convocation subcommittee, and
member of the Education Committee. He also serves as Associate Editor
(Neuro-Behavioral Pharmacology section) of the

Journal of Pharmacology and

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Experimental Therapeutics

. Finally, Dr. Buccafusco was an invited speaker and
discussant at the National Institutes on Aging symposium on Age-Related Neurobe-
havioral Research: An Integrative Cognitive Neuroscience Agenda for the 21st Cen-
tury, held in 1999.
Dr. Buccafusco’s research area includes the development of novel treatment
modalities for Alzheimer’s disease and related disorders. In 1988 his laboratory was
the first to report the cognitive enhancing action of low doses of nicotine in non-
human primates. Since that time he has studied numerous novel memory-enhancing
agents from several pharmacological classes in this model. His most recent work is
directed at the development of single molecular entities that act on multiple CNS
targets, not only to enhance cognitive function, but also to provide neuroprotection,
or to alter the disposition and metabolism of amyloid precursor protein. Dr. Bucca-
fusco also has studied the toxic effects of organophosphorus anticholinesterases used
as insecticides and as chemical warfare agents. In particular, he has studied the
behavioral/cognitive alterations associated with low level, chronic exposure to such
agents. Finally, his work in the area of drug abuse has centered around the role of
central cholinergic neurons in the development of physical dependence on opiates,
and in the expression of withdrawal symptoms. These studies have been supported

by continuous federally sponsored grants awarded by the National Institutes of
Health, the Department of Defense, and the Veterans Administration.

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Contributors

Adam C. Bartolomeo

Wyeth Neuroscience
Wyeth-Ayerst Research
Princeton, NJ

Carl A. Boast, Ph.D.

Behavioral Neuroscience
Wyeth-Ayerst Research
Princeton, NJ

Bruno Bontempi, Ph.D.

Laboratoire de Neurociences
Comportementales et Cognitives
Talence, France

Peter J. Brasted, Ph.D.

Department of Experimental

Psychology
University of Cambridge
UK

Jerry J. Buccafusco, Ph.D.

Alzheimer’s Research Center
Department of Pharmacology and
Toxicology
Medical College of Georgia and
Department of Veterans Affairs
Medical Center
Augusta, GA

Philip J. Bushnell, Ph.D.

Neurotoxicology Division
U.S. Environmental Protection
Agency
Research Triangle Park, NC

Peter Curzon

Abbott Laboratories
Abbott Park, IL

Michael W. Decker, Ph.D.

Abbott Laboratories
Abbott Park, IL

Stephen B. Dunnett, Ph.D.

MRC Cambridge Center for
Brain Repair
University Forvie Site
Cambridge, UK

Ralph L. Elkins, Ph.D.

Department of Psychiatry and
Health Behavior
Medical College of Georgia and
Medical Research Service
Department of Veterans Affairs
Medical Center
Augusta, GA

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Gerard B. Fox, Ph.D.

Abbott Laboratories
Abbott Park, IL

John H. Graham, Ph.D.

Biology Department

King College
Bristol, TN

Leonard L. Howell, Ph.D.

Department of Psychiatry and
Behavioral Sciences
Yerkes Regional Primate Center
Emory University
Atlanta, GA

William J. Jackson, Ph.D.

Department of Physiology and
Endocrinology
Medical College of Georgia
Augusta, GA

Robert Jaffard, Ph.D.

Laboratoire de Neurociences
Comportementales et Cognitives
Talence, France

John R. James, Ph.D.

Department of Pharmacy and
Pharmaceutics
School of Pharmacy
Virginia Commonwealth University

Richmond, VA

Edward D. Levin, Ph.D.

Department of Psychiatry and
Behavioral Sciences
Duke University Medical Center
Durham, NC

Frédérique Menzaghi, Ph.D

Arena Pharmaceuticals, Inc.
San Diego, CA

T. Edward Orr, Ph.D.

Department of Psychiatry and
Health Behavior
Medical College of Georgia and
Medical Research Service
Department of Veterans Affairs
Medical Center
Augusta, GA

Merle G. Paule, Ph.D.

Behavioral Toxicology Laboratory
National Center for Toxicology
Research
Jefferson, AR

Mark A. Prendergast, Ph.D.

Tobacco and Health Research
Institute
University of Kentucky
Lexington, KY

John A. Rosecrans, Ph.D.

Department of Pharmacology
Virginia Commonwealth University
Richmond, VA

Jay S. Schneider, Ph.D.

Department of Pathology
Anatomy and Cellular Biology
Thomas Jefferson University
Philadelphia, PA

Sheldon B. Sparber, Ph.D.

Department of Pharmacology
University of Minnesota
Medical School
Minneapolis, MN

Alvin V. Terry, Jr., Ph.D.

University of Georgia Clinical
Pharmacy Program Medical
College of Georgia
Augusta, GA

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Thomas J. Walsh, Ph.D.

Department of Psychology
Rutgers University
New Brunswick, NJ

Paul A. Walters

Department of Psychiatry and Health
Behavior
Medical College of Georgia and Medical
Research Service
Department of Veterans Affairs Medical
Center
Augusta, GA

Kristin M. Wilcox, Ph.D.

Division of Neuroscience
Yerkes Regional Primate
Research Center

Emory University
Atlanta, GA

Richard Young, Ph.D.

Department of Medicinal
Chemistry
School of Pharmacy
Virginia Commonwealth University
Richmond, VA

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Contents

Chapter 1

Choice of Animal Subjects in Behavioral Analysis

William J. Jackson

Chapter 2

The Behavioral Assessment of Sensorimotor Processes

in the Mouse: Acoustic Startle, Locomotor Activity, RotaRod, and
Beam Walking

Gerard B. Fox, Peter Curzon, and Michael W. Decker

Chapter 3

Fundamentals, Methodologies, and Uses of Taste
Aversion Learning

T. Edward Orr, Paul A. Walters, and Ralph L. Elkins

Chapter 4

Drug Discrimination

Richard Young, John R. James, and John A. Rosecrans

Chapter 5

Conditioned Place Preference: An Approach to Evaluating
Positive and Negative Drug-Induced Stimuli

John R. James, Richard Young, and John A. Rosecrans

Chapter 6

Intravenous Drug Self-Administration in Nonhuman
Primates

Leonard L. Howell and Kristin M. Wilcox

Chapter 7

Assessing Attention in Rats

Philip J. Bushnell

Chapter 8

Assessment of Distractibility in Non-Human Primates
Performing a Delayed Matching-to-Sample Task

Mark A. Prendergast

Chapter 9

Inhibitory Avoidance Behavior and Memory Assessment

John H. Graham and Jerry J. Buccafusco

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Chapter 10

Spatial Navigational (Water Maze) Tasks

Alvin V. Terry, Jr.

Chapter 11

The Delayed Non-Match-to-Sample Radial Arm Maze Task:
Application to Models of Alzheimer’s Disease

Carl A. Boast, Thomas J. Walsh, and Adam C. Bartolomeo

Chapter 12

Use of the Radial-Arm Maze to Assess Learning and
Memory in Rodents

Edward D. Levin

Chapter 13

An Operant Analysis of Fronto-Striatal Function in the Rat

Stephen B. Dunnett and Peter J. Brasted

Chapter 14

Use of Autoshaping with Non-Delayed and Delayed
Reinforcement for Studying Effects upon Acquisition and
Consolidation of Information

Sheldon B. Sparber

Chapter 15

Assessing Frontal Lobe Functions in Non-Human Primates

Jay S. Schneider

Chapter 16

Validation of a Behavioral Test Battery for Monkeys

Merle G. Paule

Chapter 17

Theoretical and Practical Considerations for the
Evaluation of Learning and Memory in Mice

Robert J. Jaffard, Bruno Bontempi, and Frederique Menzaghi

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1

Chapter

Choice of Animal Subjects
in Behavioral Analysis

William J. Jackson

Contents

I. Introduction
II. Origin of the Albino Laboratory Rat
III. The Laboratory Rat in Behavioral Research
IV. Advantages of Rat Models
V. Disadvantages of Rat Models
VI. Strain Selection
A. The Wistar Rat Colony
B. The Long-Evans Strain
C. Strains from Columbia University
D. Sprague-Dawley Rats
E. Holtzman Rats
F. N/Nih Rats
G. Wild Norway Rats
VII. Inbred Rat Strains Selected for Various Behavioral Traits
A. Rat Strains Selected for Preference of — and
Sensitivity to —Alcohol
B. ACI Strain
C. Strains Bred for Various Serotonin Receptors
D. Roman Strain
E. Maudsley Strains
F. Tryon’s Maze-Bright and Maze-Dull Rats
G. Spontaneous Hypertensive Rats
H. Flinders Sensitive Line and Flinders Resistant Line
I. Dahl Salt Sensitive Rats

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2

Methods of Behavior Analysis in Neuroscience

VIII. Comparison of Various Rat Strains for Behavioral Characteristics
IX. Mice in Behavioral Research
X. Pigeons and Other Species Performing Traditional Non-human
Primate Tasks
XI. Non-Human Primates
A. Advantages
B. Disadvantages
C. Commonly Used Non-human Primates in Biomedical
Research
D. Basic Behavioral Differences Among Monkey Species
E. Primate Cognitive Skills
F. Transfer of Training
XII. Discussion
References

I. Introduction

Many researchers using behavioral techniques are not primarily interested in animal
behavior, as such. Typically, behavioral animal research in physiology and pharma-
cology is designed to provide a model for human processes, and great effort is given
toward the development of animal models that reflect behavioral processes shared
by animals and humans.

1

Whenever using animals as research subjects, behavioral
physiologists, pharmacologists, and geneticists commonly describe their work as
animal models of human characteristics, or justify their work on the basis of rele-
vance to human pathology. The search for treatment and cure of illness with behav-
ioral implications will continue to lean heavily upon animal models. Animal subjects
will assist in evaluation of the effectiveness of putative treatments, and in providing
further insight into underlying physiologic mechanisms of human pathology. Sub-
sequent chapters of this book focus on many of these models. Anxiety, addiction,
taste-aversion, attention deficit, and disorders of learning and memory are examples
of behavioral disorders that are often studied

via

animal models.
Behavioral researchers usually plan most aspects of their research projects to
fine detail, but may fail to give the same level of planning toward selection of the
species that is to be used as a model. The goal of this chapter is to provide a glance
at some of the most popular species used for behavioral neuroscientific research.
Most of the discussion is given to rats and non-human primates, but there is a section
on mice and notes about other species as well.

II. Origin of the Albino Laboratory Rat

Contemporary strains of albino laboratory rats were bred from captured wild Norway
rats. The wild Norway rat is believed to have originated from temperate regions of

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Choice of Animal Subjects in Behavioral Analysis

3

Asia and southern Russia. As civilization developed, these animals found a suitable
ecological niche in the castoffs and trash heaps of man and, as economic pests, spread
rapidly over the world. Norway rats were common throughout Europe and the British
Isles by the early 18th century. By 1775, Norway rats were common in the northeastern
U.S.

2

Because the large number of rats represented an economic hazard, special breeds
of rat-catching terrier dogs were bred. In a roundabout way, the breeding of these dogs
was responsible for the beginnings of the albino laboratory rat.
The recorded speculation of early breeders of albino strains was that they were
products of the “sport” of rat baiting, which was outlawed by decree.

3

Rat baiting
involved the release of 100 or 200 newly trapped rats into a fighting pit. A trained
terrier dog was then put into the pit, and a measurement of the time until the last
rat was killed was taken. Wagers were placed on the speed of the various dogs. Rat
baiting required that many rats be trapped and retained in pounds. Historical records
relate that albinos were removed from these pounds and kept for show purposes and
breeding. Many albino show rats were tamed and offspring were selected for docility
and color. Because the captured wild Norway rats were fierce and difficult to handle,
the more docile albinos were the stock from which early European laboratory

researchers selected their first animals.

III. The Laboratory Rat in Behavioral Research

After serving for a hundred years as a subject in behavioral and physiological
research experiments, the albino rat is a generally accepted model. However, early
researchers had to justify their selection of animals, as opposed to humans, and it
would be valuable for us to remember their rationale. Memories of the initial battles
to gain acceptance for animal models of human cognition have dimmed over time;
today, studies using animals are commonly accepted.
Behavioral investigation using rats as subjects in the U.S. arose from work at
Clark University Biological Laboratory during the 1890s. According to Miles,

4

Stewart was working with wild gray rats to determine the effects of alcohol, diet,
and barometric change on the activity of the animals as early as 1894. The feral-
captured gray rats were fierce and difficult to handle, and Stewart was forced to
switch to the more docile white rats by 1895. The rats were trained to run through
the maze to earn food reward. Kline

5

invented several problem boxes that served as
prototypes for contemporary devices still used to measure cognition and learn-
ing/memory in rodents. Small used a maze patterned after the garden maze found
at Hampton Court Palace in England to measure observable behavior that would
indicate learning by the rats. Use of the white rat as a research subject in behavioral
research was given a great impetus by the investigations of Watson

6

at Chicago
University. Watson’s ideas firmly cemented the white rat as a fixture of experimental
studies in behavior.

7

At the same time of Stewart’s behavioral work in the 1890s, Donaldson was
using the white rat at the University of Chicago for anatomical and physiological
research. Donaldson’s rat colony became the parent stock at the Wistar Institute of

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4

Methods of Behavior Analysis in Neuroscience

Anatomy and Biology in Philadelphia. In 1924, a book entitled

The Rat

14

by H. H.
Donaldson gave impetus to the white rat as an acceptable research subject and
provided wisdom about the use of animal subjects in biomedical research. In one
comment from

The Rat

, Donaldson stated that “in enumerating the qualifications of
the rat as a laboratory animal, and in pointing out some of its similarities to man,
it is not intended to convey the notion that the rat is a bewitched prince or that man
is an overgrown rat, but merely to emphasize the accepted view that the similarities
between mammals having the same food habits tend to be close, and that in some
instances, at least, by the use of equivalent ages, the results obtained with one form
can be very precisely transferred to the other.”

IV. Advantages of Rat Models

Rats are commonly employed as animal subjects in contemporary medical research,
and general acceptance of the white rat as an animal research subject has increased
in synchrony with an increased appreciation of the value of behavioral research.
Since rats are small, clean, relatively inexpensive, easily handled and maintained,
widely available, have short twenty-one day gestational periods, and a short two to
three year lifespan, their use as research subjects offers many advantages. These
advantages are amplified in application to research problems that require large
numbers of animals. Likewise, the relatively short twenty-one day gestation and
approximate three year lifespan of the rat provide a practical opportunity to study
the stages of development and aging.

V. Disadvantages of Rat Models

Despite the numerous advantages offered by the laboratory rat model, there are
difficulties that must be considered. First, it is more difficult to draw parallels
between rodent and human behavior and physiology, than to compare non-human
primates with humans. The behavioral characteristics of the rodent subject are more

primitive, making behavioral comparison more complicated. Second, it is more
difficult to establish stimulus control of the rodent’s behavior in training paradigms.
Often, it is necessary to use aversive electrical shock or drastic food deprivation to
motivate the rodent subjects. These severe control procedures further complicate the
comparison with humans, since such control measures are unacceptable for human
research.

VI. Strain Selection

Once it has been decided to use rats as subjects in a given research endeavor, the
question of strain selection becomes important. There are many outbred and inbred

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Choice of Animal Subjects in Behavioral Analysis

5

strains of rats available on the commercial market, and the effects of many common
treatments differ according to the strain selected. Early in my scientific training I
received a lesson about this important fact by finding that hippocampectomy in Spra-
gue-Dawley rats increased several forms of activity, and increased one type of error
in a Lashley III maze. However, when the same hippocampectomy was effected in
Fischer strain rats, the animals consistently became less active, and did not make the
same Lashley III error. Normally, Fisher rats are much less active than normal Sprague-
Dawley rats, and the hippocampectomy may have removed cortical inhibition of
behavioral tendencies that were already present in the normal unoperated controls.
There are many published papers that show differences among strains of laboratory

rats regarding the effects of various treatments, shown in the following sections.

A. The Wistar Rat Colony

The first rats brought from Chicago to the Wistar Institute in Philadelphia by Donald-
son in 1906 became the parent stock of a rat colony, whose offspring were sold to
research facilities throughout the U.S. and many other countries until 1960. The
Wistar Institute was a leader in determining laboratory animal husbandry practices
necessary to support a large rat colony. By 1922, the colony had a total population
of about 6000 rats. The commercial rights to the sale of the Wistar Rat were sold
in 1960.
From the beginning, the Wistar Institute maintained a random bred, heteroge-
neous colony. Therefore, there was considerable variability in the commercial colony
maintained by the Institute. It is unknown whether albino lines other than those
provided by Donaldson were introduced into the Wistar Colony. According to Lind-
sey,

2

it is documented that outside breeders were brought into the colony to boost
breeding production.
Most of the albino laboratory rats used in the U.S. are linked to the colony of
the Wistar Institute. In previous discussion, the role of Donaldson in the development
of the albino laboratory rat model was mentioned. Donaldson’s rat colony became
the parent stock at the Wistar Institute of Anatomy and Biology. Even Donaldson
himself did not know whether stock from the European labs found its way to the
U.S., or whether the first albinos in the U.S. were derived from wild rats captured
in the U.S.

8,9

B. The Long-Evans Strain

Prior to 1920, two members of the faculty at the University of California at Berkley,
J. A. Long and H. M. Evans, were interested in the estrous cycle of the rat. To
support their research interests, Long and Evans established one of the leading strains
of rats that continues to bear their names. The origins of the Long-Evans rat colony
were described in a monograph entitled

The Oestrous Cycle in the Rat and Its
Associated Phenomena

. In that monograph, it was stated that the colony descended
from a cross made around 1915 “between several white females and a wild gray

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6

Methods of Behavior Analysis in Neuroscience

male” that was trapped along the bank of Strawberry Creek, which ran through the
university campus. Dr. Leslie Bennett, a colleague of Dr. Long, is quoted as saying
that the white females were supplied by the Wistar Institute.

2

The Long-Evans rats exhibited varied fur color. The coats represented in the

colony are black, gray, and hooded. The hooded animals are characterized by pig-
mented fur on their heads and often along the spine. These animals have a pigmented
iris, and their visual acuity far exceeds that of the albino strains.

C. Strains from Columbia University

The Crocker Institute of Cancer Research at Columbia University began in 1913 to
inbreed six major bloodlines of rats to be used in cancer research. Researchers at
the institute had noticed that certain rats were more susceptible to sarcomas induced
by feeding tapeworm eggs. The inbreeding program was designed to determine if
the susceptibility to sarcoma was genetic.

2

Dr. Maynie R. Curtis was the chief
developer of the inbred rat colony. Dr. Curtis purchased a few breeding pairs of rats
from each of four local breeders whose names were August, Fischer, Marshall, and
Zimmerman. The obtained rats had different coloring, and these external character-
istics were used as markers to help identify the various strains. The Marshall rats
were albinos. The Fischer and Zimmerman rats were non-agouti piebalds, but did
carry the albino gene. The August rats were the most varied, and included some
with pink eyes. In 1941, a group of rats with red eyes were obtained from a breed
in Connecticut. These animals were seed stock for brother-sister mating and are
progenitors of several popular inbred strains. The first litter of pedigreed rats at
Columbia were from mating number 344, and were the first representatives of the
Fischer 344 strain.

D. Sprague-Dawley Rats

There appears to be little record of the origin of the Sprague-Dawley strain. The

primary stock is believed to have been established by Robert W. Dawley, who was
a physical chemist at the University of Wisconsin. Mr. Dawley included his wife’s
maiden name, which was Sprague, to name the rats. Mr. Dawley later established
Sprague-Dawley, Inc. to advance the commercial sale of his rats. Lindsey

2

cites a
letter from Mr. Dawley to the National Institutes of Health (NIH), dated July of
1946, in which he states that the original parents were a hybrid hooded male rat of
exceptional size, and vigor, that was genetically half albino. He was mated to a
white female, and subsequently to his white female offspring for seven generations.
The origin of the hooded male is unknown, but the first white female is believed to
be from the Wistar colony. Selection was on the basis of many factors, including
high lactation, rapid growth, vigor, good temperament, and high resistance to arsenic
trioxide. The original company continues today under the name Harlan Sprague-
Dawley.

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Choice of Animal Subjects in Behavioral Analysis

7

There are many sub-lines of the Sprague-Dawley animals, and they are often
used in behavioral research. They are randomly bred strains, but differ from one
another. The Sprague-Dawley strain is also sold by the Charles River Co. There is
variability in the stock marketed by the different suppliers. For example, Pollock

and Rekito

10

found that the Sprague-Dawley rats marketed by Harlan differed from
Sprague-Dawley rats marketed by the Charles River Co. in regard to hypertensive
response to chronic L-NAME-induced nitric oxide synthase inhibition.

E. Holtzman Rats

A major sub-line of the Sprague-Dawley line is the Holtzman rat. These rats are
provided by a company established by E. C. Holtzman, who was a former employee
of the Sprague-Dawley Co. Sprague-Dawley animals were the original seed stock
of the Holtzman line.

F. N/Nih Rats

The National Institutes of Health (NIH), through systematic interbreeding of eight
inbred rat strains, created a heterogeneous stock of rats.

11
This strain, called N/Nih,
has been maintained by a strict breeding policy to ensure that mating pairs of
subsequent generations are distantly related. Predictably, the large genetic variability
among the N/Nih rats results in varied phenotypes. Several selective breeding pro-
grams that originated with N/Nih rats have developed strains of inbred rats with
characteristics of great interest to behavioral research. These inbred strains include
rats chosen for high and low alcohol consumption and sensitivity. The genealogy
of the animals maintained by the NIH can be found on their web site
( or by correspondence

with them.

G. Wild Norway Rats

Not many researchers are hardy enough to explore the use of wild rats as animal
subjects, although some have.

12,13

Discussion of the laboratory rat would be incomplete
without some mention of how the typical albino laboratory rat differs from the original
wild stock. Albino laboratory rats were originally selected for docility, i.e., a reduced
tendency to flee from humans or to struggle and bite when handled. There are impli-
cations of this selection for docility. For example, novel objects that induce avoidance
or fear in wild rats often elicit approach or apparent curiosity in the albino laboratory
strains. Albino males do not attack other male rats with the intensity typical of wild
male rats. The food preferences of the wild rats also differ from the albino strains.

12,14

Differences in behavior are paralleled by changes in growth and in the relative weight
of the adrenal glands. Wild rats are genetically heterogeneous, and thus there is
considerable behavioral variation among wild rats. Barnett

15

used the techniques of

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Methods of Behavior Analysis in Neuroscience

ethology to observe the innate behavioral characteristics of wild Norway rats in groups.
Under these conditions, it is obvious that rats have a complex social structure that is
not generally measured in typical laboratory testing. Barnett also describes rat behavior
that most of us have casually noticed, but generally have not understood. Barnett
explained and illustrated the role of many body postures and gestures in rat society.
Barnett has also reviewed techniques for testing wild Norway rats.

15

VII. Inbred Rat Strains Selected for Various
Behavioral Traits

With the explosion of interest in the genetic basis of physiology and behavior, there
are many papers in which various inbred strains of rats are compared to outbred
strains according to the criteria of interest.



Populations of outbred rats such as the
Wistar, and hetereogenous strains such as the N/Nih rats, manifest considerable
variability along almost any behavioral or physiological attribute. Selective breeding
programs for high or low manifestations of various phenotypes have resulted in
inbred strains that are useful for many areas of behavioral research.
The names of these strains do not follow a systematic nomenclature. One attempt

to standardize the rat strains

16

recommended that the rat nomenclature system follow
that of inbred mice. The following quote from Festing and Staats presents the problem:
“In many cases a strain name has been changed whenever a strain has been transferred
to a new laboratory, and in other cases strains which have only a distant relationship
have been given the same name. This is particularly the case with strains descended
from Wistar outbred stock, which tend to be named ‘WIS’ or some other name
beginning with W.” The nomenclature system recommended by Festing and Staats has
not been universally followed. Many strains are named after the university or some
other prominent, but not obvious, feature of their development. The following sections
describe basic aspects of some common inbred strains used in behavioral research.

16

A. Rat Strains Selected for Preference of — and Sensitivity
to — Alcohol

Normal out-bred (e.g., Wistar) and heterogeneous rats (e.g., N/Nih) do not typically
drink much alcohol, but there are large individual differences in alcohol drinking
over a large population of these rats. By selectively breeding for high or low alcohol
intake and high and low neurosensitivity, lines of rats that exhibit these characteristics
regarding alcohol have been derived. The inbred alcohol-preferring rats typically
consume up to ten times the amount of alcohol taken by normal out-bred or heter-
ogeneous rats. The selectively bred rat lines include the alcohol-preferring (P),
alcohol-accepting (Alko Alcohol — AA), Sardinian alcohol-preferring, and high
alcohol drinking (HAD) rats.

17

These lines do not share all other behavioral traits.

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Choice of Animal Subjects in Behavioral Analysis

9

Higher than normal alcohol drinking has also been observed in several groups
of rats that were selectively bred for other specific behavioral traits. These include
the Tryon Maze-Bright and Tryon Maze-Dull rats,

18

the Roman High- and Low-
Avoidance rats,

19

and the Fawn-Hooded rats that have serotonin receptor abnormal-
ity.

20,21

Typically, the ACI strain of rats will not voluntarily drink alcohol.

B. ACI Strain

The ACI line was originated by Curtis and Dunning at the Columbia University
Institute for Cancer Research. Initially, the primary phenotype of interest was sus-
ceptibility to estrogen-induced tumors; however, there are also a number of behav-
ioral differences associated with this strain. For example, the Brh sub-line shows
low defecation response and high activity response in the open field test.

C. Strains Bred for Various Serotonin Receptors

Selective inbreeding of N/Nih rats has resulted in strains that vary in sensitivity to
5-HT

1A

receptor stimulation. Overstreet

21

has established a selective breeding pro-
gram for high (HDS) and low (LDS) sensitivity to the hypothermic response of the
5-HT

1A

agonist 8-OH-DPAT. These two rat lines are believed to differ in behavioral
tests of depression, but not of anxiety. The lines also differ in post-synaptic 5-HT

1A

receptors. Pre-synaptic mechanisms are not affected.

D. Roman Strain

The Roman strain of rats was selectively bred from Wistar stock for high and low
performance in two-way active avoidance learning.

22

RHA/Verh rats acquire active
avoidance (shuttle box) performance quickly, because they are less emotionally
reactive, but more active in regard to locomotion. RLA/Verh rats cope with the active
avoidance problem more passively, and become immobile when faced with the
avoidance task. They show increased defecation in the open field and increased
activity in the hypothalamic-pituitary-adrenal axis. The two lines of the Roman strain
differ in many respects at the behavioral and neurochemical level (for review).

19

E. Maudsley Strains

The Maudsley Reactive (MR) rats were selectively bred for high defecation in the
open field test.

23

Maudsley Non-Reactive rats (MNRA) were selectively bred for
low rates of defecation in the open field test. The strains were genetically selected
from outbred Wistar progenitors.

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